In 1966, Chambon and co-workers discovered a 116 kD enzyme which was characterized in detail in subsequent years and is now called PARP (EC 2.4.2.30) (poly(adenosine-5′-diphosphoribose) polymerase), PARS (poly(adenosine-5′-diphosphoribose) synthase) or ADPRT (adenosine-5′-diphosphoribose transferase). In the plant kingdom (Arabidopsis thaliana) a 72 kD (637 amino acids) PARP was found in 1995 (Lepiniec L. et al., FEBS Lett 1995; 364(2): 103-8). It was not clear whether this shorter form of PARP is a plant-specific individuality or an artefact (“splice” variant or the like). The 116 kD PARP enzyme has to date been unique in animals and in man in its activity, which is described below. It is referred to as PARP1 below to avoid ambiguity.
The primary physiological function of PARP 1 appears to be its involvement in a complex repair mechanism which cells have developed to repair DNA strand breaks. The primary cellular response to a DNA strand break appears moreover to consist of PARP1-catalyzed synthesis of poly(ADP-ribose) from NAD+ (cf. De Murcia, G. et al. (1994) TIBS, 19, 172).
PARP 1 has a modular molecular structure. Three main functional elements have been identified to date: an N-terminal 46 kD DNA binding domain; a central 22 kD automodification domain to which poly(ADP-ribose) becomes attached, with the PARP 1 enzyme activity decreasing with increasing elongation; and a C-terminal 54 kD NAD5 binding domain. A leucine zipper region has been found within the automodification domain, indicating possible
protein-protein interactions, only in the PARP from Drosophila. All PARPs known to date are presumably active as homodimers.
The high degree of organization of the molecule is reflected in the strong conservation of the amino acid sequence. Thus, 62% conservation of the amino acid sequence has been found for PARP 1
from humans, mice, cattle and chickens. There are greater structural differences from the PARP from Drosophila. The individual domains themselves in turn have clusters of increased conservation. Thus, the DNA binding region contains two so-called zinc fingers as subdomains (comprising motifs of the type CX3CX28/30HX2C), which are involved in the Zn2+-dependent recognition of DNA single strand breaks or single-stranded DNA overhangs (e.g., at the chromosome ends, the telomeres). The C-terminal catalytic domain comprises a block of about 50 amino acids (residues 859-908), which is about 100% conserved among vertebrates (PARP “signature”). This block binds the natural substrate NAD* and thus governs the synthesis of poly(ADP-ribose) (cf. de Murcia, loc. cit.). The GX3GKG motif in particular is characteristic of PARPs in this block.
The beneficial function described above contrasts with a pathological one in numerous diseases (stroke, myocardial infarct, sepsis etc.). PARP is involved in cell death resulting from ischemia of the brain (Choi, D. W., (1997) Nature Medicine, 3, 10, 1073), of the myocardium (Zingarelli, B., et al (1997), Cardiovascular Research, 36, 205) and of the eye (Lam, T. T. (1997), Res. Comm. in Molecular Pathology and Pharmacology, 95, 3, 241).
PARP activation induced by inflammatory mediators has also been observed in septic shock (Szabo, C., et al. (1997), Journal of Clinical Investigation, 100, 3, 723). In these cases, activation of PARP is accompanied by extensive consumption of NAD+. Since four moles of ATP are consumed for the biosynthesis of one mole of NAD+, the cellular energy supply decreases drastically. The consequence is cell death.
PARP1 inhibitors described in the abovementioned specialist literature are nicotinamide and 3-aminobenzamide. 3,4-Di-hydro-5-[4-(1-piperidinyl)butoxyl-1(2H)-isoquinolone is disclosed by Takahashi, K., et al (1997), Journal of Cerebral Blood Flow and Metabolism 17, 1137. Further inhibitors are described, for example, in Banasik, M., et al. (1992) J. Biol. Chem., 267, 3, 1569 and Griffin, R. J., et al. (1995), Anti-Cancer Drug Design, 10, 507.
High molecular weight binding partners described for human PARP1 include the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) which binds via a zinc finger motif and a BRCT (BRCA1 C-terminus) module (amino acids 372-524) (Masson, M., et al., (1998) Molecular and Cellular Biology, 18, 6, 3563).